Avian infectious coryza caused by infection with A.pg is known as important respiratory diseases in poultry. Poultry suffering from avian infectious coryza have a running nose, swelling of the face and epiphora as cardinal symptoms. Avian infectious coryza brings about a great economical damage since it leads to decrease in the breeding rate of poultry, retarding of egg laying, decrease in egg production or failure of egg laying.
Page et al. classified A.pg into three serotypes A, B and C (Non-patent reference 1) whereas Sawata et al. classified into two serotypes 1 and 2 (Non-patent reference 2). Subsequently, Kume et al. reported that serotype A by Page corresponds to serotype 1 by Sawata et al. whereas serotype C by Page corresponds to serotype 2 by Sawata et al. (Non-patent references 3 and 4). Nowadays, serotype A (serotype 1) of A.pg (hereinafter also refer to as “A.pg-A”) and serotype C (serotype 2) of A.pg (hereinafter also refer to as “A.pg-C”) are considered to be a main causative agent of avian infectious coryza.
For protection from avian infectious coryza, an inactivated vaccine has hitherto been used widely which is obtained by inactivating the cells of A.pg-A or A.pg-C with formalin, thimerosal, and the like. However, adverse side effects caused by such an inactivated vaccine has been an issue as it was reported that local necrotic lesions are formed in the inoculated chicken when the vaccine is administered (Non-patent reference 5), and hence, development of a highly safe vaccine is earnestly desired.
Under the circumstances, there have been developed or are under development a component vaccine where only a protective antigen, i.e. an effective component, obtained from bacterial cells or culture supernatant is used; a recombinant vaccine where a gene coding for a protective antigen is cloned by genetic recombination technique and expressed in bacteria, yeast, animal cells, plant cells, insect cells, and the like, and a product expressed in a large amount is purified and used; and a vector vaccine where a gene coding for a protective antigen is inserted into a viral vector, and the like.
For instance, Tokunaga et al. have successfully purified, from culture of A.pg-A, a polypeptide having about 130 kd of molecular weight from said A.pg-A, said polypeptide inducing production of a hemagglutination-inhibition antibody (HI antibody) and protecting against avian infectious coryza by A.pg-A (Patent reference 1). Furthermore, they have cloned a DNA fragment coding for said 130 Kd polypeptide and expressed said gene fragment in E. coli to find that the produced polypeptide could protect from avian infectious coryza caused by Avibacterium paragallinarum serotype A. Besides, they used said DNA fragment coding for the 130 Kd polypeptide as a probe to identify an HMT p210 gene coding for serotype A HMT p210 polypeptide, an outer-membrane protein having a hemagglutination activity, consisting of 2,042 amino acids. They also cloned from Avibacterium paragallinarum serotype C a DNA fragment hybridizable with said DNA fragment to obtain serotype C HMT p210 gene (Patent reference 2). They compared nucleotide sequences of open reading frame of HMT p210 genes of Avibacterium paragallinarum serotypes A and C to report that homology between both genes was about 80% as a whole, and that a region of about 3.4 kbp at the 5′-end (hereinafter refer to as “Region 1”) and a region of about 1.2 kbp at the 3′-end (hereinafter refer to as “Region 3”) had a very high homology while a region of about 1.5 kbp flanked by the two regions (hereinafter refer to as “Region 2”) had a low homology (Patent reference 2).
It was also reported by Noro et al. that the HMT p210 gene discovered by Tokunaga et al. is important for a target region of a serotype specific vaccine. Noro et al. reported that, by immunizing poultry with a peptide encoded by a DNA fragment of from 4,801 bp to 5,157 bp, which is a portion of HMT p210 gene coding for the A.pg-A HMT p210 polypeptide, said peptide induced an HI antibody and had a vaccinal effect against A.pg-A (Patent reference 3). Noro et al. also reported in the 143rd Meeting of the Japanese Society of Veterinary Science held on Apr. 3-5, 2007, Japan that, by immunizing poultry with a peptide encoded by a DNA fragment of 5.5 kbp, which is a portion of HMT p210 gene coding for the A.pg-C HMT p210 polypeptide, said peptide induced an HI antibody and had a vaccinal effect against A.pg-C.
Serum diagnosis has not been performed for avian infectious coryza since, in addition to acute progress of the disease, poultry infected with A.pg are not likely to induce an antibody even after onset of the disease. On the other hand, poultry undergone vaccination induce an antibody to Hemagglutinin (hereinafter also referred to as “HA”) on the surface of the A.pg cells and, for estimation of vaccinal effect in vitro, a hemagglutination-inhibition test (hereinafter also referred to as “HI test”) with an anti-HA antibody has been performed. HI test, where fresh chicken erythrocytes or chicken erythrocytes fixed with glutaraldehyde are used, is indicated to have defects: (1) since fresh chicken erythrocytes are necessary for estimation of vaccinal effect of A.pg-A, it is troublesome and laborsome such as obtaining chickens for blood (breeding and managing chickens under condition of separation from pathogens), bleeding, blood treatment, and the like, (2) stable results are not likely to be obtained since the results may be influenced by the lots of chickens erythrocytes and estimation of an antibody titer is made by subjectivity of a person who measures.
On the other hand, Sun et al. reported, for alternative serum diagnostic of HI test, blocking ELISA (B-ELISA) using a serotype specific monoclonal antibody (Non-patent reference 6). B-ELISA is ELISA where serotype A or C cells disrupted by sonication were used as an antigen and monoclonal antibodies reactive with the respective serotypes were used to competitively detect antibodies in sera. This method is advantageous in that it has a higher sensitivity than HI test and may treat multiple antibodies. However, it requires four steps, i.e. addition of sera, addition of monoclonal antibodies, addition of anti-mouse IgG-HRP labeled antibody and addition of a substrate for development, one more step than in ordinary ELISA, rendering troublesome procedures. Also, for this method, serotype specific monoclonal antibodies need be obtained. For manufacturing a kit, it will take trouble of preparing and adding a plate immobilized with an antigen and reference serum as well as monoclonal antibodies. Besides, it is noted that said B-ELISA is a system that detects an antibody to only an antigenic epitope recognized by a single monoclonal antibody for the respective serotypes. With a system that detects an antibody to a single epitope, however, when said epitope is lost due to mutation of A.pg, it is highly liable not to detect its infection or an antibody induced by vaccination.    Patent reference 1: Japanese Patent Publication No. 10-84969    Patent reference 2: WO98/12331    Patent reference 3: Japanese Patent Publication No. 2005-218414    Non-patent reference 1: Am. J. Vet. Res., 23:85-95, 1962    Non-patent reference 2: Jpn. J. Vet. Sci., 40:645-652, 1978    Non-patent reference 3: Am. J. Vet. Res., 41:757-760, 1980    Non-patent reference 4: Am. J. Vet. Res., 41:1901-1904, 1980    Non-patent reference 5: Avian Dis., 15:109-117, 1971    Non-patent reference 6: Int. Ass. Bio. (IABS), 35:317-320, 2007